help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wu, L.-W.
Right arrow Articles by Lau, K.-H. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, L.-W.
Right arrow Articles by Lau, K.-H. W.
The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 4 1126-1135
Copyright © 1997 by The Endocrine Society

Endocrinological Oncology

Fluoride at Mitogenic Doses Induces a Sustained Activation of p44mapk, but Not p42mapk, in Human TE85 Osteosarcoma Cells1

Li-Wha Wu, Hyun Koo Yoon, David J. Baylink, Lee M. Graves and K.-H. William Lau

Departments of Medicine and Biochemistry, Loma Linda University, and the Mineral Metabolism Unit, Jerry L. Pettis Memorial Veterans Administration Medical Center, Loma Linda, California 92357; and the Department of Pharmacology, University of North Carolina (L.M.G.), Chapel Hill, North Carolina 27514

Address all correspondence and requests for reprints to: Dr. K.-H. William Lau, Mineral Metabolism (151), Jerry L. Pettis Memorial Veterans Administration Medical Center, 11201 Benton Street, Loma Linda, California 92357. E-mail: LAUB{at}LLVAMC.VA.GOV


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fluoride, at micromolar concentrations, stimulates bone cell proliferation in vitro. In this study, we sought to test whether fluoride at mitogenic doses increases the tyrosyl phosphorylation level and specific activity of a mitogen-activated protein kinase (MAPK) in human TE85 osteosarcoma cells. Analysis by immunoprecipitation with antiphosphotyrosine antibody followed by Western analysis using an anti-pan extracellular signal-regulated kinase antibody revealed that fluoride at the optimal mitogenic dose (i.e. 100 µmol/L) induced a time-dependent increase in the steady state tyrosyl phosphorylation level of p44mapk, but not p42mapk, with the maximal increase (4- to 13-fold) after 1–3 h fluoride treatment. The effect was sustained in that a 9-fold increase was seen after 12 h of the fluoride treatment. The sustained nature of the effect is consistent with an inhibition of dephosphorylation rather than a direct stimulation of phosphorylation. The fluoride effect on the tyrosyl phosphorylation level of p44mapk was dose dependent, with the optimal dose being 100 µmol/L fluoride. The mitogenic dose of fluoride also increased the specific activity and the in-gel kinase activity of p44mapk, but not that of p42mapk, in a time-dependent manner similar to the effect on the p44mapk tyrosyl phosphorylation level. Fluoride at the same micromolar doses did not increase cell proliferation, tyrosyl phosphorylation, or specific activity of any MAPK in human skin foreskin fibroblasts, which are fluoride-nonresponsive cells. Consistent with the interpretation that the effect of fluoride on the steady state tyrosyl phosphorylation level of p44mapk is a consequence of an inhibition of a phosphotyrosyl phosphatase (PTP), mitogenic doses of orthovanadate, a bone cell mitogen and a PTP inhibitor, also increased the steady state tyrosyl phosphorylation level of p44mapk, but not p42mapk, in a time-dependent sustained manner similar to that observed with fluoride. Together, these findings support the concept that inhibition of a PTP activity in bone cells could lead to an activation of MAPK activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FLUORIDE IS an effective agent to increase spinal bone density in osteoporotic patients (1, 2). Bone morphometric studies showed that the osteogenic action of fluoride is mediated by an increase in the number of osteoblasts (3, 4). Farley and co-workers (5) have shown that fluoride in vitro stimulated the proliferation and differentiation of bone cells in monolayer cultures, indicating a direct anabolic action of fluoride on bone cells. The mitogenic effect of fluoride was subsequently confirmed by several laboratories in bone cells of various species, including humans (6, 7, 8, 9, 10, 11). The optimal dose of fluoride to stimulate bone cell proliferation and differentiation in vitro is low, between 10–100 µmol/L (depending on the type of culture medium used) (5, 6), which corresponds to the serum fluoride concentration in fluoride-treated patients (12). The mitogenic action of fluoride appears to be bone cell specific (5, 6, 13).

The molecular mechanism by which fluoride stimulates bone cell proliferation is unknown. Although fluoride at millimolar concentrations is a potent activator of adenylyl cyclase through stimulation of Gs, fluoride at the mitogenic, micromolar concentrations did not have an effect on cellular cAMP production in bone cells (13). Recent evidence suggests that the mitogenic action of fluoride could be mediated through a signal transduction pathway involving tyrosyl phosphorylation (13, 14). Our past work suggests that the osteogenic action of fluoride could be mediated at least in part by increasing the steady state tyrosyl phosphorylation of key signaling proteins through inhibition of an osteoblastic fluoride-sensitive phosphotyrosyl phosphatase (PTP) (13). Our hypothesis is supported by several pieces of circumstantial evidence: 1) fluoride at micromolar concentrations specifically inhibited an osteoblastic PTP, and the apparent Ki for fluoride inhibition was within the mitogenic fluoride dose range for bone cells (5, 6, 15); 2) fluoride treatment increased the net protein tyrosyl phosphorylation level in intact bone cells and isolated cell membrane (13); 3) other PTP inhibitors of this fluoride-sensitive PTP, i.e. vanadate and molybdate, also stimulated bone cell proliferation at doses that inhibited this fluoride-sensitive PTP activity (13, 16); 4) treatment with human TE85 osteosarcoma cells with mitogenic doses of fluoride for 24 h significantly increased the steady state tyrosyl phosphorylation level of at least 13 cellular proteins in human bone cells (17); and 5) the apparent molecular masses of some of these proteins were similar to those of some of the known signaling molecules, including mitogen-activated protein kinase (MAPK) (17).

MAPKs consist of a large family of serine/threonine protein kinases that are activated by the dual phosphorylation on both a tyrosine and a threonine residue (18, 19). In mammals, the extracellular signal-regulated kinase (ERK) subgroup of MAPK has been examined in detail. They are found to be rapidly phosphorylated and activated in response to mitogenic and differentiating stimuli in many different cell types (20, 21, 22, 23, 24). Two major isoforms, p44mapk (or ERK1) and p42mapk (or ERK2), are ubiquitously expressed in mammalian species and are highly conserved across species (25, 26). Although the physiological role of each MAPK (or ERK) has not been established, MAPKs are thought to play a pivotal role in integrating and transducing extracellular signals required for growth and differentiation (18, 19, 20, 21, 22, 23, 24). Accordingly, we postulate that the mitogenic action of fluoride may involve an increase in the tyrosyl phosphorylation and the corresponding activation of one or both ERK/MAPKs in human bone cells.

In this study, we examined whether fluoride at mitogenic, micromolar doses would increase the steady state tyrosyl phosphorylation level and the specific activity of ERK/MAPKs in human bone cells. Human osteoblast-like TE85 osteosarcoma cells were chosen for these studies because previous studies demonstrated that these cells reproducibly responded to the mitogenic action of fluoride in vitro (27). In addition, as an indirect test of whether the effect of fluoride on tyrosyl phosphorylation would be consistent with an inhibition of PTP, we compared the time-dependent effect of fluoride on MAPK phosphorylation to that of orthovanadate, which is a known PTP inhibitor (28) and a bone cell mitogen (16).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Tissue culture supplies were obtained from Falcon (Oxnard, CA). DMEM, bovine calf serum, and trypsin were purchased from Life Technologies (Grand Island, NY). BSA (fraction V, RIA grade) was purchased from U.S. Biochemical Corp. (Cleveland, OH). Ammonium persulfate, and bromophenol blue were products of ICN Biomedicals (Costa Mesa, CA). [{gamma}-32P]ATP (7000 Ci/mol) was obtained from DuPont-New England Nuclear (Boston, MA). Polyclonal antiphosphotyrosine (anti-PY) antibody was purchased from Promega (Los Angeles, CA). Monoclonal anti-pan ERK antibody was a product of Transduction Laboratories (Lexington, KY). Enhanced chemiluminescence (ECL) detection system was obtained from Amersham Corp. (Arlington Heights, IL). Phenylmethylsulfonylfluoride, leupeptin, aprotonin, pepstatin, NaF, calmidizolium, glycine, ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), ethylenediamine tetraacetate (EDTA), dithiothreitol (DTT), HEPES, Nonidet P-40, Tris, Triton X-100, MgCl2, Folin-Ciocalteu’s phenol reagent, NaVO4, sodium molybdate, para-nitrophenyl phosphate, ß-glycerophosphate, and myelin basic protein (MBP) were products of Sigma Chemical Co. (St. Louis, MO). SDS was from National Diagnostics (Atlanta, GA). Prestained molecular size protein standards were obtained from Bio-Rad Laboratories (Hercules, CA). The Mono Q anion exchange column was obtained from Pharmacia LKB Biotechnology (Uppsala, Sweden). Other reagents were of reagent grade and were purchased from Fisher Chemical Co. (Los Angeles, CA) or Sigma Chemical Co.

Cell cultures

Human TE85 osteosarcoma cells were originally obtained from Dr. J. Fogh of Sloan-Kettering Institute (New York, NY). These cells exhibit osteoblastic characteristics, e.g. responding to PTH by increasing cAMP production (29) and to 1,25-dihydroxyvitamin D3 by increasing the syntheses of collagen and the osteoblast-specific protein, osteocalcin (30). Normal human skin fibroblasts were prepared from a foreskin sample by collagenase digestion (6). Both human cell lines were maintained in DMEM supplemented with 10% bovine serum. Cells at passages 2–7 were used in this study.

[3H]Thymidine incorporation assay

Human TE85 cells or foreskin fibroblasts were seeded at 2000 cells/cm2 in 24-well dishes in DMEM supplemented with 10% bovine calf serum for 24 h. The cells were rinsed once with DMEM and serum deprived in DMEM supplemented with 0.1% BSA for 16–24 h before the addition of effectors. Effectors were diluted in DMEM supplemented with 0.1% BSA. Growth-arrested (i.e. serum deprived) cells were treated with effectors (e.g. 0–500 µmol/L NaF or 0–5 µmol/L NaVO4) or corresponding vehicle control for 24 h. DNA synthesis, an index for cell proliferation, was measured by stimulation of [3H]thymidine incorporation (1.5 µCi/well) during the final 2 h of incubation (31). Although this assay does not measure cell proliferation directly, we have previously shown that stimulation of [3H]thymidine incorporation reflected an increase in bone cell number (32). This assumption was supported by the finding that fluoride and FBS (1%) stimulated [3H]thymidine incorporation in bone cells with a corresponding increase in cell number (13).

Determination of steady state tyrosyl phosphorylation level of cellular proteins

TE85 cells were plated in DMEM supplemented with 10% bovine calf serum at a density of 2500 cells/cm2 in 100-mm culture dishes. After plating for 2 days, the cells were rinsed with fresh DMEM and cultured in DMEM containing 0.05% bovine calf serum for 18–24 h before the addition of effectors.

To measure the steady state tyrosyl phosphorylation level of intracellular proteins, the cells were rinsed once with DMEM and incubated with fresh DMEM for 1 h. NaF (at the indicated doses) or the vehicle control (DMEM supplemented with 0.1% BSA) was added, and the cells were incubated for 3 h at 37 C. The cells were then washed twice with ice-cold phosphate-buffered saline containing 1 mmol/L NaVO4 and lysed with a lysis buffer containing 50 mmol/L Tris-HCl (pH 7.5), 0.1% Triton X-100, 137 mmol/L NaCl, 2 mmol/L EDTA, 1 mmol/L NaVO4, 1 mmol/L phenylmethylsulfonylfluoride, 10 µg/mL leupeptin, and 10 µg/mL aprotinin. The protein concentration in each lysate, after trichloroacetic acid precipitation, was determined according to the method of Lowry et al. (33). Equal amounts of cellular proteins from each treatment group were separated on a 10% SDS-PAGE column, and the phosphotyrosyl proteins were identified with Western blots using the anti-PY antibody followed by the ECL detection assay. The relative density of the cellular protein bands with an apparent molecular mass similar to that of MAPK (i.e. 42–44 kDa) was measured by scanning laser densitometry, and the results were reported as a percentage of corresponding vehicle-treated control value.

Determination of steady state tyrosyl phosphorylation level of MAPK

To measure the level of tyrosyl-phosphorylated MAPKs, TE85 cells were treated with the indicated concentration of effectors for the indicated period of time as described above. After treatment, cells were lysed, and equal amounts of cellular proteins were immunoprecipitated with the anti-PY antibody (diluted 1:200). The immunoprecipitation procedure was adapted from that described by Argetsinger et al. (34). The immune complexes were subjected to SDS-PAGE followed by Western analysis using the anti-pan ERK (MAPK) antibody (1:1000 dilution). The anti-pan ERK recognizes all members of the ERK subset of the MAPKs. The MAPKs were visualized with the ECL detection system. The relative level of the tyrosyl-phosphorylated MAPKs was quantified by scanning laser densitometry. Because immunoprecipitation should isolate only the tyrosyl-phosphorylated MAPK species, and because the MAPKs each contain only a single tyrosyl phosphorylation site, this assay approach should allow measurements of the relative steady state tyrosyl phosphorylation level of MAPKs.

Measurement of the MAPK protein level

The effector-treated or vehicle-treated cells were lysed as described above. Equal amounts of cellular protein from each treatment group were immunoprecipitated using the anti-pan ERK antibody (1:125 dilution). The immune complexes were resolved by SDS-PAGE, the MAPK/ERKs were identified by Western analysis with the anti-pan ERK antibody (1:1000 dilution) and the ECL detection system, and the MAPK/ERK protein level was quantitated with scanning laser densitometry.

Assay for p44mapk and p42mapk specific activities using MBP as the substrate

The vehicle- and effector-treated cells were lysed with an extraction buffer [50 mmol/L Tris-HCl (pH 7.5), 1% Nonidet P-40, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.2 mmol/L NaVO4, 10 mmol/L NaF, 1 mmol/L sodium molybdate, 0.5 µg/mL leupeptin, 10 µg/mL aprotinin, and 0.7 µg/mL pepstatin]. The cell lysate, after being filtered through a 0.2-µm filter, was subjected to fast protein liquid chromatography (FPLC) separation on a Mono Q column. The column was first washed with 5 mL of a buffer containing 20 mmol/L HEPES (pH 7.4), 20 mmol/L ß-glycerophosphate, 2 mmol/L DTT, 2 mmol/L EDTA, and 2 mmol/L EGTA, then eluted with a 25-mL linear gradient of 0–0.5 mol/L NaCl in the same buffer at a flow rate of 1 mL/min. MAPK activity in each fraction (1 mL) was assayed with MBP as previously described (35). Briefly, 12.5-µL aliquots of each fraction were incubated with MBP (0.3 mg/mL) for 20 min at 30 C in a final volume of 25 µL containing 25 mmol/L ß-glycerophosphate (pH 7.3), 1.25 mmol/L EGTA, 1 mmol/L DTT, 10 mmol/L MgCl2, 0.15 mmol/L NaVO4, 2 µmol/L protein kinase inhibitor peptide, 10 µmol/L calmidizolium, 1 mg/mL BSA, and 100 µmol/L [{gamma}-32P]ATP (SA, 2000 cpm/pmol). The reaction was terminated by spotting 20 µL of the reaction mixture onto a 2 x 2-cm Whatman P-81 paper square (Whatman, Clifton, NJ). The unbound radioactivity was removed by extensive washing with 150 mmol/L phosphoric acid, followed by a final wash with absolute ethanol. The air-dried paper squares were counted in a liquid scintillation counter. The elution profile of p42mapk and p44mapk was confirmed by immunoblotting the MAPK isoforms with the anti-pan ERK antibody. The p42mapk and p44mapk peaks were then individually pooled, and the pooled fractions were reassayed for total p42mapk and p44mapk activities. The protein content in each fraction was determined by the Folin-Lowry protein assay (33). The p42mapk and p44mapk activities were normalized against the protein content.

In-gel MBP kinase assay

The in-gel MBP kinase assay, modified from those of Kameshita and Fujiawa (36) and Gotoh et al. (37), was used to confirm the effects of fluoride on MAPK activity. Briefly, the fluoride- or vehicle-treated TE85 cells were lysed with the extraction buffer as described above. Equal amounts (10 µg) of cellular protein (without boiling the sample before SDS-PAGE) were separated on a 10% SDS-PAGE containing 0.5 mg/mL MBP. The gel was washed twice with 50 mmol/L Tris-HCl, pH 8.0, and 20% 2-propanol for 30 min each time with vigorous shaking at room temperature to remove SDS, and then washed once with buffer A (50 mmol/L Tris-HCl, pH 8.0, and 5 mmol/L ß-mercaptoethanol) for 1 h with vigorous shaking at room temperature to remove 2-propanol. The proteins in the gel were denatured by washing the gel twice (30 min each) with buffer A containing 6 mol/L guanidine HCl at room temperature and renatured in buffer A with 0.04% Tween-40 with vigorous shaking for 16–24 h at 4 C with at least five changes of buffer. The in-gel phosphorylation reaction was carried out at room temperature by incubating the gel in a reaction mixture consisting of 40 mmol/L HEPES (pH 8.0), 2 mmol/L DTT, 0.1 mmol/L EGTA, 5 mmol/L MgCl2, 25 µmol/L ATP, and 250 µCi [{gamma}-32P]ATP for 1 h with vigorous shaking. The reaction was terminated by washing the gel 10 times with 5% trichloroacetic acid and 1% sodium pyrophosphate for 2 h each time until the solution did not contain a significant amount of radioactivity. The resulting gel was dried and subjected to autoradiography with an intensifier at -70 C overnight. The relative phosphorylation level was determined with a scanning laser densitometry.

Statistical analysis

The statistical significance of the differences in [3H]thymidine incorporation and MAPK specific activity between groups was analyzed by two-tailed Student’s t test and ANOVA. The difference was considered significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of fluoride on [3H]thymidine incorporation in human TE85 osteosarcoma cells

Figure 1Go shows that fluoride in the micromolar range significantly and reproducibly stimulated [3H]thymidine incorporation by 20–60% above the control value in a dose-dependent, biphasic manner with the optimal mitogenic dose of approximately 100 µmol/L. These findings confirm previous findings (27, 29, 30) that fluoride is mitogenic to human TE85 osteosarcoma cells.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 1. Fluoride at micromolar doses stimulates [3H]thymidine incorporation into DNA in human TE85 cells. [3H]thymidine incorporation is shown as a percentage of the control value. The dashed line represents 100% of the control value (1450 ± 335 cpm). Bovine calf serum (1%) increased [3H]thymidine incorporation in this experiment by 355 ± 32% (i.e. 5147 ± 464 vs. 1450 ± 335 cpm; P < 0.001). Results are shown as the mean ± SD of six replicates. *, P < 0.01; **, P < 0.05 (by two-tailed Student’s t test; ANOVA indicates a significant stimulation at P < 0.05). This experiment was repeated at least five times.

 
Effect of mitogenic doses of fluoride on the steady state tyrosyl phosphorylation level of cellular proteins in TE85 osteosarcoma cells

Western immunoblot analysis with a commercial anti-PY polyclonal antibody showed that treatment of TE85 cells with 0–500 µmol/L fluoride for 3 h increased the steady state tyrosyl phosphorylation level of several cellular proteins; one of which had an apparent molecular mass of 44 kDa (Fig. 2Go). Scanning laser densitometric measurements of the 44-kDa protein indicated that fluoride at the effective doses (i.e. between 50–200 µmol/L) reproducibly increased the steady state tyrosyl phosphorylation level of the 44-kDa protein. The stimulatory effect was dose dependent, with the maximal stimulation seen at approximately 100 µmol/L fluoride, which was also the optimal dose that stimulated TE85 cell proliferation (Fig. 1Go).



View larger version (71K):
[in this window]
[in a new window]
  		Figure 2. Mitogenic doses of fluoride increase the steady state tyrosyl phosphorylation of a cellular protein with an apparent molecular mass of 44 kDa in human TE85 cells. Quiescent TE85 cells were treated with vehicle or 25–500 µmol/L fluoride for 3 h. Molecular mass standards are shown on the left. The arrow indicates the protein band with an apparent molecular mass of 44 kDa. Similar results were obtained in two separate experiments.

 
Effect of fluoride on the steady state tyrosyl phosphorylation level of p44mapk

To test whether the p44 protein was a MAPK, an anti-pan ERK antibody was used in the Western blot analysis after immunoprecipitation with the anti-PY antibody to identify the tyrosyl-phosphorylated MAPKs. The 44-kDa protein reacted strongly with the anti-pan ERK antibody, indicating that the 44-kDa protein was the p44mapk. Figure 3Go shows that treatment of TE85 cells with 100 µmol/L fluoride caused a time-dependent increase in the steady state tyrosyl phosphorylation level of p44mapk. In this experiment, the steady state tyrosyl phosphorylation of p44mapk was increased after 1–3 h of fluoride treatment. The increase was maximally induced by 4- to 13-fold after 12 h of fluoride treatment. The time-dependent stimulation of tyrosyl phosphorylation of p44mapk was highly reproducible (i.e. seen in each of the three repeat experiments), but the time required for the maximal stimulation varied from experiment to experiment (i.e. from 3–12 h of fluoride treatment). The fluoride-induced increase was sustained: a 2–9 fold increase in the tyrosyl phosphorylation level of p44mapk was detectable even after 12 h of fluoride treatment. In contrast, there was no detectable amount of tyrosyl-phosphorylated p42mapk in TE85 cells, and the fluoride treatment had no notable effect on the steady state tyrosyl phosphorylation level of p42mapk.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 3. Fluoride at a mitogenic dose increases steady state tyrosyl phosphorylation of p44mapk in a time-dependent manner. Quiescent TE85 cells were stimulated with 100 µmol/L fluoride for the indicated time. Molecular mass standards are shown on the left. Arrows indicate the position of p44mapk. The top panel shows fluoride-treated cells, and the bottom panel represents vehicle-treated control cells. Similar results were obtained in three separate experiments.

 
It should also be noted that there appeared to be a small, time-dependent increase in the steady state tyrosyl phosphorylation of p44mapk in the vehicle-treated cells (Fig. 3Go). However, because human bone cells are known to secrete growth factors into conditioned medium, and because many growth factors are known to increase tyrosyl phosphorylation, it is speculated that this small, time-dependent increase may simply reflect the time-dependent accumulation of growth factors in the conditioned medium.

Fluoride at the test doses had no significant stimulatory effect on the steady state cellular level of p44mapk protein throughout the 12-h treatment period (Fig. 4Go). [Fluoride at these doses under the same conditions also did not significantly affect the cellular level of p42mapk (data not shown).] These findings indicate that the increase in the steady state tyrosyl phosphorylation level of p44mapk was probably not the result of an increased de novo synthesis of p44mapk.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 4. Fluoride at a mitogenic dose has no stimulatory effect on de novo p44mapk synthesis. Quiescent TE85 cells were treated with 100 µmol/L fluoride for the indicated time. Equal amounts of each cell lysate proteins were immunoprecipitated with the anti-pan ERK, blotted onto a nitrocellulose membrane, probed with the anti-pan ERK antibody, and detected with ECL. Arrows indicate the position of p44mapk. This experiment was repeated once with the same result.

 
Dose-dependent effect of fluoride on the tyrosyl phosphorylation level of p44mapk

Figure 5Go reveals that 1-h treatment with fluoride (10–500 µmol/L) increased the steady state tyrosyl phosphorylation level of p44mapk (2- to 3-fold), but not that of p42mapk. The stimulation was dose dependent and biphasic, with a maximal stimulatory dose of 100–200 µmol/L fluoride, similar to that stimulated the cell proliferation (Fig. 1Go). The dose-dependent stimulatory effect of fluoride on TE85 cells was reproducible and was seen in all three repeat experiments.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. Dose-dependent increase in steady state tyrosyl phosphorylation of p44mapk by fluoride. Quiescent TE85 cells were stimulated with vehicle or 10–500 µmol/L fluoride for 1 h. The position of the p42 protein is indicated on the left. The arrow on the right denotes the position of p44mapk. Similar results were obtained in three repeat experiments.

 
Effects of fluoride on proliferation and MAPK tyrosyl phosphorylation in normal human foreskin fibroblasts

Previous studies (5, 6, 13) indicated that the mitogenic action of micromolar concentrations of fluoride appeared to be specific for bone cells and did not stimulate the proliferation of skin fibroblasts. Thus, we investigated whether the stimulatory effects of the same test doses (10–500 µmol/L) of fluoride would increase steady state tyrosyl phosphorylation level of MAPK in normal human foreskin fibroblasts, which are fluoride nonresponsive cells. Figure 6AGo shows that fluoride at micromolar (10–500 µmol/L) doses had no significant stimulatory effect on [3H]thymidine incorporation into the DNA of human skin fibroblasts. Similar to the lack of mitogenic activity, treatment of human foreskin fibroblasts with 100 µmol/L fluoride for 0–12 h also did not lead to any detectable change in either p44mapk or p42mapk isoenzymes (Fig. 6BGo). Likewise, incubation with the test dose (10–500 µmol/L) of fluoride did not show a dose-dependent effect on the tyrosyl phosphorylation of either p44mapk or p42mapk in human foreskin fibroblasts (Fig. 6BGo).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. Lack of a stimulatory effect of fluoride on [3H]thymidine incorporation (A) and MAPK tyrosyl phosphorylation (B) in normal human foreskin fibroblasts. In A, the dashed line represents 100% of the vehicle control value (109 ± 21 cpm). Bovine calf serum (10%) increased [3H]thymidine incorporation in this experiment by 360 ± 82%. Results are shown as the mean ± SD of six replicates. ANOVA indicates that there was no statistically significant effect of fluoride on [3H]thymidine incorporation in these human skin fibroblasts. B shows the time-dependent (upper lane) and dose-dependent (lower lane) effects, after 1 h of incubation, of fluoride on the steady state tyrosyl phosphorylation levels of p42mapk and p44mapk in normal human foreskin fibroblasts. Experiments in both A and B were repeated at least three times, and no significant or consistent dose- or time-dependent effect of fluoride on the tyrosyl phosphorylation level of either p42mapk or p44mapk was found.

 
Effect of mitogenic doses of fluoride on the specific activity of MAPKs

Tyrosyl phosphorylation of MAPK is required for the activation of its kinase activity. Therefore, the effects of mitogenic doses of fluoride on the specific activities of MAPKs in TE85 cells were measured. The FPLC Mono Q anion exchange chromatography was employed to separate the major MAPKs isoenzymes for analysis. Figure 7Go shows that this chromatographic approach separated the bound MAPKs into two major peaks of MBP kinase activity. Immunoblotting analysis revealed that the first peak was predominately p42mapk, whereas the second peak was predominately p44mapk (Fig. 7Go, inset). Figure 8Go indicates that fluoride at 100 µmol/L significantly stimulated the specific activity of p44mapk, without an effect on the specific activity of p42mapk. The specific activity was maximally induced (2- to 3-fold) after 3 h of fluoride treatment and was sustained even after 10 h of fluoride incubation.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 7. Mono Q FPLC separation of p42mapk and p44mapk in human TE85 osteosarcoma cell extracts. Human TE85 cells, treated with the vehicle control or with 100 µmol/L fluoride for 24 h, were extracted with the lysis buffer and then separated by FPLC using a Mono Q (HR 5/5) anionic column. The identities of p42mapk (peak I) and p44mapk (peak II) were confirmed by Western immunoblot (shown in the inset).

 


View larger version (29K):
[in this window]
[in a new window]
  		Figure 8. Fluoride increases the specific MBP kinase activity of p44mapk, but not that of p42mapk, in human TE85 cells in a sustained manner. Growth-arrested TE85 cells were stimulated with 100 µmol/L fluoride for the indicated times. Cell lysates were prepared and subjected to Mono Q FPLC separation. The p42mapk and p44mapk peaks were separately pooled and assayed. The left panel shows the time-dependent effect of fluoride on p42mapk, and the right panel illustrates the time-dependent activation of p44mapk. The results are shown as the mean ± SD (n = 3). The results of this experiment were reproduced in another experiment.

 
To confirm that fluoride at a mitogenic dose would indeed activate p44mapk activity in TE85 cells, an in-gel MAPK assay was performed. Figure 9AGo shows the autoradiograph of the dose-dependent (25–500 µmol/L) effect of fluoride on the in-gel MBP kinase activity of both p44mapk and p42mapk. Treatment with fluoride at 25–200 µmol/L for 1 h consistently (in each of the repeat experiments) stimulated the in-gel MBP kinase activity of p44mapk. Scanning laser densitometric measurement of the autoradiographs revealed that maximal stimulation (2- to 3-fold) was seen with approximately 100 µmol/L fluoride (Fig. 9BGo). In contrast, fluoride at these test doses had no consistent effect on the in-gel MBP kinase activity of the p42mapk. Figure 10Go shows that the stimulatory effect of fluoride was time dependent. It was maximally induced (2- to 3-fold) after 3 h of fluoride treatment and was sustained even after 6 h of fluoride incubation. Thus, these findings are consistent with the premise that an increase in the tyrosyl phosphorylation level would lead to activation of p44mapk in TE85 osteosarcoma cells.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 9. Fluoride increases the MBP kinase activity of p44mapk in a dose-dependent manner in the in-gel MBP kinase assay. A, Autoradiography. The positions of p44mapk and p42mapk, respectively, are indicated by the arrows on the left. B, Laser densitometric measurements of the MBP kinase activities of p42mapk and p44mapk. These results were repeated in two separate studies.

 


View larger version (35K):
[in this window]
[in a new window]
  		Figure 10. Fluoride increases the MBP kinase activity of p44mapk in a time-dependent manner in the in-gel MBP kinase assay. A, Autoradiograph of the time-dependent effects of 100 µmol/L fluoride on MBP kinase activities. The positions of p44mapk and p42mapk, respectively, are indicated by arrows on the left. B, Laser densitometric measurements of the MBP kinase activities of p42mapk and p44mapk. These results were reproduced in a separate study.

 
Effect of mitogenic doses of orthovanadate on the steady state tyrosyl phosphorylation level of p44mapk in TE85 osteosarcoma cells

To indirectly test the hypothesis that the effect of fluoride on the steady state tyrosyl phosphorylation level of p44mapk is mediated by an inhibition of the fluoride-sensitive PTP in osteoblasts, we determined the time-dependent effect of a known PTP inhibitor and bone cell mitogen, NaVO4, on the steady state tyrosyl phosphorylation level of p44mapk in TE85 cells. We reasoned that if fluoride acts to increase the phosphorylation level of p44mapk by inhibition of the dephosphorylation, the time course of fluoride on the steady state p44mapk tyrosyl phosphorylation would be similar to that of NaVO4 at the mitogenic doses.

Consistent with our previous findings (13, 16), NaVO4 significantly (P < 0.01) stimulated [3H]thymidine incorporation (i.e. 20–60% above the control value) in human TE85 osteosarcoma cells, with the optimal dose being between 1–2 µmol/L (data not shown). Figure 11Go shows the time-dependent effect of 1 µmol/L NaVO4 on the steady state tyrosyl phosphorylation level of the MAPKs. An increase in the steady state tyrosyl phosphorylation of p44mapk was first detected after 30 min of vanadate treatment. Like fluoride, this dose of NaVO4 had no detectable effect on the p42mapk tyrosyl phosphorylation level. The effect of fluoride on the p44mapk tyrosyl phosphorylation level was time dependent, and the maximal increase (up to 14-fold) was observed between 6–12 h of the treatment. Accordingly, similar to the effect of fluoride, a mitogenic dose of NaVO4 induced a sustained stimulation on p44mapk tyrosyl phosphorylation.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 11. Orthovanadate at a mitogenic dose stimulates p44mapk tyrosyl phosphorylation in human TE85 osteosarcoma cells in a sustained fashion. The top panelshows cells treated with 1 µmol/L orthovanadate for the indicated length of time, and the bottom panel represents the corresponding vehicle-treated cells. The arrow indicates the position of p44mapk.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we present compelling evidence that treatment with fluoride in vitro at the micromolar concentrations that stimulated human bone cell proliferation markedly increased the steady state tyrosyl phosphorylation level and activated the MBP kinase activity of p44mapk (ERK1) in human TE85 osteosarcoma cells. The optimal dose for the fluoride-induced increase in steady state tyrosyl phosphorylation and that for the activation of the kinase activity of p44mapk were each approximately 100 µmol/L fluoride, which was also the optimal dose of fluoride to stimulate the proliferation of these bone cells.

Inasmuch as the physiological significance of these findings remains unclear, there is a large body of evidence supporting a key role for MAPK activation in the cell proliferation process. The most compelling of the evidence is that antisense messenger ribonucleic acid of p44mapk and p42mapk or overexpression of the p44mapk mutants in fibroblasts completely blocked the growth factor-mediated cell proliferation (38). Thus, the finding that the fluoride-mediated stimulation of human bone cell proliferation and the fluoride-dependent activation of p44mapk exhibited similar dose-dependent curves with the same optimal dose raises an interesting possibility that the fluoride-dependent activation of p44mapk might be associated with the fluoride-mediated stimulation of human bone cell proliferation. Two pieces of circumstantial evidence in this study support this speculation: 1) orthovanadate, a stimulator of human bone cell proliferation, also increased the steady state tyrosyl phosphorylation level of p44mapk with a dose-dependent curve similar to that of stimulation of human bone cell proliferation; and 2) fluoride at the same micromolar doses had no stimulatory effect on the proliferation or the tyrosyl phosphorylation level of MAPKs in normal human foreskin fibroblasts, which are fluoride nonresponsive cells (5, 6, 13). Accordingly, we should not overlook the possibility that the activation of p44mapk may play an essential role in the stimulation of bone cell proliferation by fluoride or vanadate.

It is intriguing that the stimulatory effect of fluoride and orthovanadate appears to be specific for p44mapk, as neither effector has a stimulatory effect on p42mapk (ERK2). However, it should be noted that different signal transduction pathways may use different MAPK isoenzymes. For example, the activation of protein kinase C by vasopressin and phorbol 12-myristate 13-acetate involved the selective activation of p42mapk in vascular smooth muscle cells (39), and insulin exerted its effect in the skeletal muscle by sequential activation of p44mapk and p42mapk (40). It has also been reported that different transcription factors are phosphorylated and activated by p42mapk and p44mapk, respectively (41). Accordingly, it can be speculated that fluoride acts to stimulate bone cell proliferation by the selective activation of the signal transduction pathway(s) that involves p44mapk as opposed to those mediated by p42mapk.

An important finding of this study is that the fluoride-induced increases in the steady state tyrosyl phosphorylation and activity of p44mapk are sustained in nature (i.e. even after 12 h of incubation). The time course of the fluoride-mediated stimulation is unique, in that treatment of mammalian cells with polypeptide growth factors, such as insulin in adipocytes (20, 42) and epidermal growth factor in PC12 cells (43) and bone cells (17) most often led to a rapid and transient activation of MAPK, which usually peaked 2–10 min after the treatment. Thus, these findings suggest that fluoride acts through a different mechanism than the polypeptide growth factors to increase the level of tyrosyl phosphorylation and the activity of the MAPKs.

The unique nature of the sustained stimulation of p44mapk phosphorylation and activity may be relevant to the molecular mechanism by which fluoride stimulates p44mapk activity. MAPKs are phosphorylated and activated by the MAPK/ERK kinases (MEKs) (44, 45) and dephosphorylated (and inactivated) by a unique family of dual specificity phosphatases, MAPK phosphatases or MKPs (46). An increase in the steady state tyrosyl phosphorylation of p44mapk could be mediated by 1) activation of MEKs, 2) inhibition of MKPs, or 3) both. However, increases in protein tyrosyl kinase activities are frequently associated with the corresponding increases in PTP activities to counter the actions of the kinases (47, 48). Thus, if the increase in the steady state tyrosyl phosphorylation level was mediated by a stimulation of kinase activity, the increase would be transient, as reported with polypeptide growth factors (17, 20, 42, 43). Conversely, if the increase was caused by an inhibition of the dephosphorylation, the effect on the steady state tyrosyl phosphorylation level would be expected to be sustained, as the steady state tyrosyl phosphorylation level would remain elevated as long as the PTP inhibitor was present. Accordingly, the sustained nature of the fluoride effect on the tyrosyl phosphorylation level and the activation of p44mapk is compatible with the hypothesis that fluoride acts to increase p44mapk activity via inhibition of a fluoride-sensitive PTP (13). The finding that orthovanadate, a known PTP inhibitor, also induced a sustained increase in the steady state tyrosyl phosphorylation level of p44mapk in a manner similar to that of fluoride is entirely consistent with this interpretation. Further supporting evidence is the observation that the inhibition of MKP-1 synthesis with cycloheximide or the expression of an inactive MKP-1 mutant in COS cells led to sustained phosphorylation and activation of MAPK (46).

There is now ample evidence that MKPs may play an important regulatory role in controlling MAPK activities (46). Therefore, it is foreseeable that fluoride could stimulate p44mapk activity through an inhibition of a fluoride-sensitive MKP. Although none of the currently known MKP isoenzymes appears to be inhibited by micromolar concentrations of fluoride, one cannot completely rule out the possibility that there might exist a yet to be identified fluoride-sensitive MKP to dephosphorylate and inactivate the p44mapk in bone cells, in light of the fact that the MAPK, MEK, and MKP each exist as a large family of multigene enzymes (21, 49, 50). In this regard, we have previously identified a fluoride-sensitive PTP in osteoblasts (51, 52). Notwithstanding, we do not yet have evidence that this fluoride-sensitive PTP is a MKP, the presence of a fluoride-sensitive PTP in osteoblasts raises the exciting possibility that there may be a fluoride-sensitive MKP in osteoblasts. Alternatively, it is possible that the fluoride-sensitive PTP may act to inhibit the dephosphorylation of upstream regulators of the p44mapk, e.g. Raf, shc, rasGAP, etc., and that activation of p44mapk is an indirect result of the inhibition of dephosphorylation (and activation) of upstream activators. In support of this concept, we have recently obtained preliminary evidence that mitogenic doses of fluoride also increased the steady state tyrosyl phosphorylation level of Raf-1 and rasGAP in TE85 cells in a sustained manner similar to that seen with p44mapk (53).

It has previously been reported that the combination of millimolar concentrations of fluoride and micromolar doses of aluminum ion was able to activate p42mapk in BC3H1 myocytes and 3T3-L1 fibroblasts through activation of a Gs protein (54). Recent studies from Dr. Bonjour’s laboratory (55, 56), with the combination of 10 µmol/L aluminum ion and 1 mmol/L NaF as the effector, raised the possibility that the aluminum fluoride-mediated stimulation of the proliferation of mouse MC3T3-E1 osteoblast-like cells may involve members of the Gi protein family. Although activators of certain G proteins have been shown to activate the MAPK pathway (57, 58), and we cannot rule out the possible involvement of a G protein in the molecular mechanism of fluoride, we do not favor the hypothesis that the micromolar doses of fluoride act to increase human bone cell proliferation (and to activate p44mapk) through a G protein-dependent mechanism similar to that of the fluoroaluminate ion for the following reasons. 1) The doses necessary to stimulate human bone cell proliferation and p44mapk are very low (i.e. 10–200 µmol/L), whereas the doses required to activate Gs protein were at least 1000-fold higher (i.e. at millimolar level). 2) We have previously reported that fluoride acts through a different mechanism than aluminum ion to stimulate human bone cell proliferation (29). 3) Although the fluoroaluminate ion is a potent activator of adenylate cyclase through the activation of Gs in many cells, including human TE85 cells (unpublished observations), the mitogenic doses (i.e. 10–200 µmol/L) of fluoride have been shown to have no significant effect on cAMP production in bone cells (13, 55). 4) Activation of cAMP production (e.g. treatment with cholera toxin) or addition of dibutyryl cAMP to human bone cells would generally lead to an inhibition of bone cell proliferation (59, 60). 5) We recently obtained preliminary evidence that although pertussis toxin abolishes the mitogenic actions and effects on tyrosyl phosphorylation of both NaF and NaVO4 on human bone cells, this toxin has no effect on AlF4--associated bone cell proliferation and tyrosyl phosphorylation (60).

In conclusion, we have demonstrated that micromolar doses of fluoride stimulated human bone cell proliferation and the steady state tyrosyl phosphorylation level and the specific activity of p44mapk, presumably via inhibition of the dephosphorylation reaction in human TE85 osteosarcoma cells. These findings extend our previous model (13) and provide important insights into the molecular mechanism by which fluoride stimulates osteoblast proliferation and differentiation. Accordingly, these findings form important ground work for our continuing efforts in searching for the mechanism of the osteogenic action of fluoride.


   Acknowledgments
 
The authors thank the Medical Media staff at the Pettis Memorial V.A. Medical Center for their assistance with the preparation of the manuscript.


   Footnotes
 
1 This work was supported in part by research grants from the NIH (DE-08681 to K.-H.W.L.) and from the V.A. (to D.J.B.). Back

Received October 23, 1996.

Revised December 27, 1996.

Accepted January 7, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Riggs BL, Seeman E, Hodgson JF, Taves DR, O’Fallon WM. 1982 Effect of fluoride/calcium regimen on vertebral fracture occurrence in postmenopausal osteoporosis: comparison with conventional therapy. N Engl J Med. 306:446–450.[Abstract]
  2. Farley SMG, Wergedal JE, Smith LC, Lundy MW, Farley JR, Baylink DJ. 1987 Fluoride therapy for osteoporosis: characterization of the skeletal response by serial measurements of serum alkaline phosphatase activity. Metabolism. 36:211–218.[CrossRef][Medline]
  3. Briancon D, Meunier PJ. 1981 Treatment of osteoporosis with fluoride, calcium, and vitamin D. In: Frost HM, ed. The orthopedic clinics of North America. Philadelphia: Saunders; vol12 :629–648.
  4. Dambacher MA, Lauffenburger T, Lammel B, Haas HG. 1978 Long term effects of sodium fluoride in osteoporosis. In: Courvoisier B, Donath A, Baud E, eds. Fluoride and bone. Bern: Hans Huber; 238–241.
  5. Farley JR, Wergedal JE, Baylink DJ. 1983 Fluoride directly stimulates proliferation and alkaline phosphatase activity of bone forming cells. Science. 222:330–332.[Abstract/Free Full Text]
  6. Wergedal JE, Lau K-HW, Baylink DJ. 1988 Fluoride and bovine bone extract influence cell proliferation and phosphatase activities in human bone cell cultures. Clin Orthop Relat Res. 233:274–282.
  7. Bellows CG, Aubin JE, Heersche JNM. 1993 Differential effects of fluoride during initiation and progression of mineralization of osteoid nodules formed in vitro. J Bone Miner Res. 8:1357–1363.[Medline]
  8. Hall BK. 1987 Sodium fluoride as an initiator of osteogenesis from embryonic mesenchyme in vitro. Bone. 8:111–116.[Medline]
  9. Reed BY, Zerwekh JE, Antich PP, Pak CYC. 1993 Fluoride-stimulated [3H]thymidine uptake in a human osteoblastic osteosarcoma cell line is dependent on transforming growth factor ß. J Bone Miner Res. 8:19–25.[Medline]
  10. Modrowski D, Miravet L, Feuga M, Bannie F, Marie PJ. 1992 Effect of fluoride on bone and bone cells in ovariectomized rats. J Bone Miner Res. 7:961–969.[Medline]
  11. Khokher MA, Dandona P. 1990 Fluoride stimulates 3H-thymidine incorporation and alkaline phosphatase production by human osteoblasts. Metabolism. 39:1118–1121.[CrossRef][Medline]
  12. Baylink DJ. 1993 Serum fluoride levels. In: Favus MJ, ed. Primers on the metabolic bone diseases and disorders of mineral metabolism, 2nd ed. New York: Raven Press; 262–263.
  13. Lau K-HW, Farley JR, Freeman TK, Baylink DJ. 1989 A proposed mechanism of the mitogenic action of fluoride on bone cells: inhibition of the activity of an osteoblastic acid phosphatase. Metabolism. 38:858–868.[CrossRef][Medline]
  14. Burgener D, Bonjour J-P, Caverzasio J. 1995 Fluoride increases tyrosine kinase activity in osteoblast-like cells: regulatory role for the stimulation of cell proliferation and Pi transport across the plasma membrane. J Bone Miner Res. 10:164–171.[Medline]
  15. Wergedal JE, Lau K-HW. 1992 Human bone cells contain a fluoride sensitive acid phosphatase: evidence that this enzyme functions at neutral pH as a phosphotyrosyl protein phosphatase. Clin Biochem. 25:47–53.[CrossRef][Medline]
  16. Lau K-HW, Tanimoto H, Baylink DJ. 1988 Vanadate stimulates bone cell proliferation and bone collagen synthesis in vitro. Endocrinology. 123:2858–2867.[Abstract]
  17. Thomas AB, Hashimoto H, Baylink DJ, Lau K-HW. 1996 Fluoride at mitogenic concentrations increases the steady state phosphotyrosyl phosphorylation level of cellular proteins in human bone cells. J Clin Endocrinol Metab. 81:2570–2578.[Abstract]
  18. Cobb MH, Boulton TG, Robbins DJ. 1991 Extracellular signal-regulated kinases: ERKs in progress. Cell Regul. 2:965–978.[Medline]
  19. Davis RJ. 1994 MAPKs: new JNK expands the group. Trends Biochem Sci. 19:470–473.[CrossRef][Medline]
  20. Ray LB, Sturgill TW. 1987 Rapid stimulation by insulin of a serine/threonine kinase in 3T3–L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro. Proc Natl Acad Sci USA. 84:1502–1506.[Abstract/Free Full Text]
  21. Boulton TG, Nye SH, Robbins DJ, et al. 1991 ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell. 65:663–675.[CrossRef][Medline]
  22. Whitehurst CE, Boulton TG, Cobb MH, Geppert TD. 1992 Extracellular signal-regulated kinases in T cells. Anti-CD3 and 4ß-phorbol 12-myristate 13-acetate-induced phosphorylation and activation. J Immunol. 148:3230–3237.[Abstract]
  23. Vietor I, Schwenger P, Li W, Schlessinger J, Vilcek J. 1993 Tumor necrosis factor-induced activation and increased tyrosine phosphorylation of mitogen-activated protein (MAP) kinase in human fibroblasts. J Biol Chem. 268:18994–18999.[Abstract/Free Full Text]
  24. Bittorf T, Jaster R, Brock J. 1994 Rapid activation of the MAP kinase pathway in hematopoietic cells by erythropoietin granulocyte-macrophage colony-stimulating factor and interleukin-3. Cell Signal. 6:305–311.[CrossRef][Medline]
  25. Boulton TG, Cobb MH. 1991 Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies. Cell Regul. 2:357–371.[Medline]
  26. Owaki H, Makar R, Boulton TG, Cobb MH, Geppert TD. 1992 Extracellular signal-regulated kinases in T cells: characterization of human ERK1 and ERK2 cDNA. Biochem Biophys Res Commun. 182:1416–1422.[CrossRef][Medline]
  27. Takada J, Chevalley T, Baylink DJ, Lau K-HW. 1996 Dexamethasone enhances the osteogenic effects of fluoride in human TE85 osteosarcoma cells in vitro. Calcif Tissue Int. 58:355–361.[Medline]
  28. Gordon JA. 1991 Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol. 201:477–482.[Medline]
  29. Lau K-HW, Yoo A, Wang SP. 1991 Aluminum stimulates the proliferation and differentiation of osteoblasts in vitro by a mechanism that is different from fluoride. Mol Cell Biochem. 105:93–105.[Medline]
  30. Lau K-HW, Wang SP, Linkhart TA, Demarest KT, Baylink DJ. 1994 Picomolar norethindrone in vitro stimulates the cell proliferation and activity of a human osteosarcoma cell line, and collagen synthesis without an effect on bone resorption. J Bone Miner Res. 9:695–703.[Medline]
  31. Gospodarowicz D, Bialecki H, Greenburg G. 1978 Purification of the fibroblast growth factor activity from bovine brain. J Biol Chem. 253:3736–3743.[Abstract/Free Full Text]
  32. Lau K-HW, Lee MY, Linkhart TA, et al. 1985 A mouse tumor-derived osteolytic factor stimulates bone resorption by a mechanism involving local prostaglandins production in bone. Biochim Biophys Acta. 840:56–68.[Medline]
  33. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951 Protein measurement with the Folin phenol reagent. J Biol Chem. 193:265–275.[Free Full Text]
  34. Argetsinger LS, Campbell GS, Yang X, et al. 1993 Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase. Cell. 74:237–244.[CrossRef][Medline]
  35. Ahn NG, Weiel JE, Chan CP, Krebs EG. 1990 Identification of multiple epidermal growth factor-stimulated protein serine/threonine kinases from Swiss 3T3 cells. J Biol Chem. 265:11487–11494.[Abstract/Free Full Text]
  36. Kameshita I, Fujiawa H. 1989 A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel. Anal Biochem. 183:139–143.[CrossRef][Medline]
  37. Gotoh Y, Nishida E, Yamashita T, Hoshi M, Kawakami M, Sakai H. 1990 Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen-activated MAP kinase of fibroblastic cells. Eur J Biochem. 193:661–669.[Medline]
  38. Pages G, Lenormand P, L’Allemain G, Chambard J-C, Meloche S, Pouyssegur J. 1993 Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci USA. 90:8319–8323.[Abstract/Free Full Text]
  39. Kribben A, Wieder ED, Li X, et al. 1993 AVP-induced activation of MAP kinase in vascular smooth muscle cells is mediated through protein kinase C. Am J Physiol. 265:C939–C945.
  40. Zhou GX, Meier KE, Buse MG. 1993 Sequential activation of two mitogen activated protein (MAP) kinase isoforms in rat skeletal muscle following insulin injection. Biochem Biophys Res Commun. 197:578–584.[CrossRef][Medline]
  41. Chuang C-F, Ng S-Y. 1994 Functional divergence of the MAP kinase pathway. ERK1 and ERK2 activate specific transcription factors. FEBS Lett. 346:229–234.[CrossRef][Medline]
  42. Haystead CM, Gregory P, Shirazi A, et al. 1994 Insulin activates a novel adipocyte mitogen-activated protein kinase kinase kinase that shows rapid phasic kinetics and is distinct from c-Raf. J Biol Chem. 269:12804–12808.[Abstract/Free Full Text]
  43. Traverse S, Gomez N, Paterson H, Marshall C, Cohen P. 1992 Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor. Biochem J. 288:351–355.
  44. Ahn NG, Seger R, Krebs EG. 1992 The mitogen-activated protein kinase activator. Curr Opin Cell Biol. 4:992–999.[CrossRef][Medline]
  45. Nishida E, Gotoh Y. 1993 The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem Sci. 18:128–131.[CrossRef][Medline]
  46. Sun H, Charles CH, Lau LF, Tonks NK. 1993 MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell. 75:487–493.[CrossRef][Medline]
  47. Gentleman S, Martensen TM, Digiovanna JJ, Chader GJ. 1984 Protein tyrosine kinase and protein phosphotyrosine phosphatase in normal and psoriatic skin. Biochim Biophys Acta. 798:53–59.[Medline]
  48. Frank DA, Sartorelli AC. 1986 Regulation of protein phosphotyrosine content by changes in tyrosine kinase and protein phosphotyrosine phosphatase activities during induced granulocytic and monocytic differentiation of HL-60 leukemia cells. Biochem Biophys Res Commun. 140:440–447.[CrossRef][Medline]
  49. Wu J, Harrison JK, Dent P, Lynch KR, Weber MJ, Sturgill TW. 1993 Identification and characterization of a new mammalian mitogen-activated protein kinase kinase, MKK2. Mol Cell Biol. 13:4539–4548.[Abstract/Free Full Text]
  50. Wu J, Lau LF, Sturgill TW. 1994 Rapid deactivation of MAP kinase in PC12 cells occurs independently of induction of phosphatase MKP-1. FEBS Lett. 353:9–12.[CrossRef][Medline]
  51. Lau K-HW, Farley JR, Baylink DJ. 1985 Phosphotyroyl specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme: comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase. J Biol Chem. 260:4653–4660.[Abstract/Free Full Text]
  52. Lau K-HW, Freeman TK, Baylink DJ. 1987 Purification and characterization of a tartrate-resistant acid phosphatase that displays neutral pH phosphotyrosyl protein phosphatase activity from bovine cortical bone matrix. J Biol Chem. 262:1389–1397.[Abstract/Free Full Text]
  53. Wu L-W, Baylink DJ, Lau K-HW. 1995 Phosphotyrosyl phosphatase inhibitors stimulate human bone cell proliferation through a sustained activation of the Ras-Raf-MAPK signal transduction pathway [Abstract M399]. J Bone Miner Res. 10(Suppl 1):S352.
  54. Anderson NG, Kilgour E, Sturgill TW. 1991 Activation of mitogen-activated protein kinase in BC3H1 myocytes by fluoroaluminate. J Biol Chem. 266:10131–10135.[Abstract/Free Full Text]
  55. Caverzasio J, Imai T, Ammann P, Burgener D, Bonjour J-P. 1996 Aluminum potentiates the effect of fluoride on tyrosine phosphorylation and osteoblast replication in vitro and bone mass in vivo. J Bone Miner Res. 11:46–55.[Medline]
  56. Caverzasio J, Zhen X, Bonjour J-P. 1996 A G protein-dependent tyrosine phosphorylation process mediates the mitogenic effect of fluoride [Abstract T341]. J Bone Miner Res. 11(Suppl 1):S385.
  57. Englaro W, Rezzonico R, Durand-Clement M, Lallemand D, Ortonne J-P, Ballotti R. 1995 Mitogen-activated protein kinase pathway and AP-1 are activated during cAMP-induced melanogenesis in B-16 melanoma cells. J Biol Chem. 270:24315–24320.[Abstract/Free Full Text]
  58. Hawes BE, van Biesen T, Koch WJ, Luttrell LM, Lefkowitz RJ. 1995 Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation. J Biol Chem. 270:17148–17153.[Abstract/Free Full Text]
  59. Mohan S, Strong DD, Hilliker S, et al. 1993 Dibutyryl cyclic adenosine monophosphate differentially regulates cell proliferation in low and high alkaline phosphatase SaOS-2 human osteosarcoma cells: evidence for mediation by the insulin-like growth factor-II system. J Cell Physiol. 156:462–468.[CrossRef][Medline]
  60. Hashimoto H, Baylink DJ, Lau K-HW. 1996 Evidence that the mechanism of osteogenic actions of fluoride in human TE85 osteosarcoma cells is not mediated through AlF3 formation [Abstract 1059]. Mol Biol Cell. 7(Suppl):183a.



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
N. V. Bogatcheva, P. Wang, A. A. Birukova, A. D. Verin, and J. G. N. Garcia
Mechanism of fluoride-induced MAP kinase activation in pulmonary artery endothelial cells
Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1139 - L1145.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
E. V. Thrane, M. Refsnes, G. H. Thoresen, M. Lag, and P. E. Schwarze
Fluoride-Induced Apoptosis in Epithelial Lung Cells Involves Activation of MAP Kinases p38 and Possibly JNK
Toxicol. Sci., May 1, 2001; 61(1): 83 - 91.
[Abstract] [Full Text] [PDF]

Home page
 Hum Exp ToxicolHome page
M Refsnes, R Becher, M Lag, T Skuland, and P E Schwarze
Fluoride-induced interleukin-6 and interleukin-8 synthesis in human epithelial lung cells
Human and Experimental Toxicology, November 1, 1999; 18(11): 645 - 652.
[Abstract] [PDF]

Home page
 J. Biol. Chem.Home page
W. B. Bowler, C. J. Dixon, C. Halleux, R. Maier, G. Bilbe, W. D. Fraser, J. A. Gallagher, and R. A. Hipskind
Signaling in Human Osteoblasts by Extracellular Nucleotides. THEIR WEAK INDUCTION OF THE c-fos PROTO-ONCOGENE VIA Ca2+ MOBILIZATION IS STRONGLY POTENTIATED BY A PARATHYROID HORMONE/cAMP-DEPENDENT PROTEIN KINASE PATHWAY INDEPENDENTLY OF MITOGEN-ACTIVATED PROTEIN KINASE
J. Biol. Chem., May 14, 1999; 274(20): 14315 - 14324.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
P. A. Hulley, F. Gordon, and F. S. Hough
Inhibition of Mitogen-Activated Protein Kinase Activity and Proliferation of an Early Osteoblast Cell Line (MBA 15.4) by Dexamethasone: Role of Protein Phosphatases
Endocrinology, May 1, 1998; 139(5): 2423 - 2431.
[Abstract] [Full Text] [PDF]

This Article